Abstract: Antibiotic-resistant bacteria, such as vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), are major causes of hospital-acquired infections and are driving forces of an escalating health crisis. We will help address the burgeoning antibiotic resistance problem by leveraging the power of bacterial genetics via unbiased genetic selections to: 1) comprehensively identify protein-protein interactions in cellular pathways that result in antibiotic resistance;and 2) discover small molecules that disable these protein-protein interactions. The proposed research will jointly exploit our expertise in the development of genetic strategies and our ongoing interest in elucidating the fundamental mechanisms of bacterial antibiotic resistance. By focusing on protein-protein interactions, this research promises to: 1) reveal new insights into the underlying biology of antibiotic resistance mechanisms and their integration into the physiological processes of the bacterial host;2) define new targets (in the form of protein-protein interactions) for innovative therapeutics to treat infections caused by drug-resistant pathogens;and 3) identify novel small-molecule drug candidates with unique modes of action. Our experimental design possesses critical strategic advantages. For example, our analyses will be done within the native context of the drug-resistant bacterial host (e.g., not by in vitro screens on isolated proteins), which will enable us to capture any potential, but as yet unknown, effects of dynamic cellular processes or post-translational modifications on key protein-protein interactions. Furthermore, we will employ powerful genetic selections capable of rapidly sifting through immense libraries to reveal even rare hits that, by definition, are functional in a physiological context. Collectively, these strategies will enable the discovery of unknown, unpredictable, and novel biological insights, not accessible by conventional means, that will be exploited to discover new candidate therapeutics with efficacy against drug-resistant bacterial infections. Public Health Relevance: Antibiotic-resistant bacteria, such as vancomycin-resistant enterococci (VRE) and methicillinresistant Staphylococcus aureus (MRSA), are major causes of hospital-acquired infections and are driving forces of an escalating health crisis. This research promises to: 1) reveal new insights into the underlying biology of antibiotic resistance mechanisms that will facilitate the development of new treatments for infections caused by drug-resistant pathogens;2) define new targets for these innovative therapeutics;and 3) identify novel small-molecule drug candidates with unique modes of action.